1. Field of the Invention
The present embodiments relate to a system and method of measuring the methylation level of DNA. Some embodiments relate to a system and method of measuring methylation level of DNA with a nucleic acid array.
2. Description of the Related Art
Biomolecule methylation, such as DNA methylation is widespread and plays a critical role in the regulation of gene expression in development, differentiation and disease. Methylation in particular regions of genes, for example their promoter regions, can inhibit the expression of these genes (Baylin et al., (2000) Trends Genet, 16, 168-174.; Jones et al., (1999) Nat Genet, 21, 163-167.). Recent work has shown that the gene silencing effect of methylated regions is accomplished through the interaction of methylcytosine binding proteins with other structural compounds of the chromatin (Razin, A. (1998) Embo J, 17, 4905-4908.; Yan et al. (2001) J Mammary Gland Biol Neoplasia, 6, 183-192.), which, in turn, makes the DNA inaccessible to transcription factors through histone deacetylation and chromatin structure changes (Bestor, (1998) Nature, 393, 311-312.). Genomic imprinting in which imprinted genes are preferentially expressed from either the maternal or paternal allele also involves DNA methylation. Deregulation of imprinting has been implicated in several developmental disorders (Kumar (2000) J Biosci, 25, 213-214.; Sasaki et al., (1993) Exs, 64, 469-486.; Zhong et al., (1996) Am J Med Genet, 64, 415-419.).
In vertebrates, the DNA methylation pattern is established early in embryonic development and in general the distribution of 5-methylcytosine (5mC) along the chromosome is maintained during the life span of the organism (Razin et al., (1993) Exs, 64, 343-357.; Reik et al., (2001) Science, 293, 1089-1093.). Stable transcriptional silencing is critical for normal development, and is associated with several epigenetic modifications. If methylation patterns are not properly established or maintained, various disorders like mental retardation, immune deficiency and sporadic or inherited cancers may follow. The study of methylation is particularly pertinent to cancer research as molecular alterations during malignancy may result from a local hypermethylation of tumor suppressor genes, along with a genome wide demethylation (Schulz (1998) Int J Oncol, 13, 151-167.).
The initiation and the maintenance of the inactive X-chromosome in female eutherians were found to depend on methylation (Goto et al., (1998) Microbiol Mol Biol Rev, 62, 362-378.). Rett syndrome (RTT) is an X-linked dominant disease caused by mutation of MeCP2 gene, which is further complicated by X-chromosome inactivation (XCI) pattern. The current model predicts that MeCP2 represses transcription by binding methylated CpG residues and mediating chromatin remodeling (Dragich et al., (2000) Hum Mol Genet, 9, 2365-2375.).
It has become a major challenge in epidemiological genetics to relate a biological function (e.g. a disease) not only to the genotypes of specific genes but also to the potential differential expression levels of each allele of the genes. DNA methylation data can provide valuable information, in addition to the genotype. It has been a goal to provide methods to determine this information, e.g. if 0, or 1 or 2 chromosomes are methylated at particular genomic locations.
DNA methylation pattern changes at certain genes often alter their expression, which could lead to cancer metastasis, for example. Thus, studies of methylation pattern in selected, staged tumor samples compared to matched normal tissues from the same patient offers a novel approach to identify unique molecular markers for cancer classification. Monitoring global changes in methylation pattern has been applied to molecular classification in breast cancer (Huang et al., (1999) Hum Mol Genet, 8, 459-470.). In addition, many studies have identified a few specific methylation patterns in tumor suppressor genes (for example, p16, a cyclin-dependent kinase inhibitor) in certain human cancer types (Herman et al., (1995) Cancer Res, 55, 4525-4530.; Otterson et al., (1995) Oncogene, 11, 1211-1216.).
Restriction landmark genomic scanning (RLGS) profiling of methylation pattern of 1184 CpG islands in 98 primary human tumors revealed that the total number of methylated sites is variable between and in some cases within different tumor types, suggesting there may be methylation subtypes within tumors having similar histology (Costello et al., (2000) Nat Genet, 24, 132-138.). Aberrant methylation of a proportion of these genes correlates with loss of gene expression.
Since methylation detection can use genomic DNA, it offers advantages in both the availability of the source materials and ease of performing the assays. Also, the methylation assay can be complementary to those used for RNA-based gene expression profiling. Typically, the use of different assays in combination is more accurate and robust for disease classification and prediction than the use of only one assay.
Accordingly, there is a need for methods of evaluating methylation levels measured across a large number of loci in a genome and within the genomes of large number of individuals. The methods and compositions described herein satisfy this need and provide other advantages as well.